NT2 cells are a clonal line of human teratocarcinoma cells that exhibit N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity after terminal differentiation into NT2-N neurons. In this study, we used modulation of glutamate excitotoxicity to characterize the pharmacological properties and specific antibodies to determine the individual subunits of NMDA receptors expressed by NT2-N neurons. The glycine site antagonist 7-chlorokynurenic acid completely blocked glutamate toxicity in a dose-dependent manner. Histamine and the polyamine agonists spermine and spermidine enhanced glutamate toxicity in a dose-dependent manner consistent with expression of an NR1-NR2B combination of subunits. The efficacy of polyamine agonists suggests the expression of one or more splice variants of the NR1 subunit that lack the putative surface loop encoded by exon 5. Surprisingly, the putative inverse agonists diaminodecane and diaminododecane also enhanced toxicity in a dose-dependent manner. The antagonists arcaine and ifenprodil completely blocked glutamate toxicity in NT2-N cells. The atypical antagonist ifenprodil inhibited toxicity with a uniformly high affinity characteristic of interaction with the NR1-NR2B combination of subunits. Expression of both NR1 and NR2 subunits were detected by Western blot analysis. Neither protein was detectable in undifferentiated cells. In contrast, 70-fold lower levels of the NR2A subunit were detected in both differentiated and undifferentiated cells. The pharmacological and immunological results indicate that a functional NR1-NR2B combination of subunits is expressed by NT2-N neurons. Despite the immunological detection of NR2A subunit, no functional combination of NR1 and NR2A subunits could be demonstrated.